In animal lipid metabolism, there are certain fatty acids, termed “essential” fatty acids, which must be supplied from vegetable sources. The essential fatty acids are required as structural components for the lipid content of cell membranes but cannot be synthesized by the animal, as summarized in Ohlrogge, et al., The Plant Cell 7: 957 (1895). This includes the essential fatty acid c9,c12-linoleic acid. Structural variants of 9,12-linoleic acid, some of which are naturally occurring, include the conjugated isomers.
The biological activity associated with conjugated linoleic acids (termed CLA) is diverse and complex. At present, very little is known about the mechanisms of action, although several preclinical and clinical studies in progress are likely to shed new light on the physiological and biochemical modes of action. The anticarcinogenic properties of CLA have been well documented. Administration of CLA inhibits rat mammary tumorigenesis, as demonstrated by Ha, et al., Cancer Res., 52: 2035s (1992). Ha, et al., Cancer Res., 50: 1097 (1990) reported similar results in a mouse forestomach neoplasia model. CLA has also been identified as a strong cytotoxic agent against target human melanoma, colorectal and breast cancer cells in vitro. A recent major review article confirms the conclusions drawn from individual studies. See Ip, Am. J. Clin. Nutr., 66 (6 Supp): 1523s (1997).
Although the mechanisms of CLA action are still obscure, there is evidence that some component(s) of the immune system may be involved, at least in vivo. U.S. Pat. No. 5,585,400 (Cook, et al.) discloses a method for attenuating allergic reactions in animals mediated by type I or TgE hypersensitivity by administering a diet containing CLA. CLA in concentrations of about 0.1 to 1.0 percent was also shown to be an effective adjuvant in preserving white blood cells. U.S. Pat. No. 5,674,901 (Cook, et al.) disclosed that oral or parenteral administration of CLA in either free acid or salt form resulted in elevation in CD-4 and CD-8 lymphocyte subpopulations associated with cell-mediated immunity. Adverse effects arising from pretreatment with exogenous tumor necrosis factor could be alleviated indirectly by elevation or maintenance of levels of CD-4 and CD-8 cells in animals to which CLA was administered. Finally, U.S. Pat. No. 5,430,066 describes the effect of CLA in preventing weight loss and anorexia by immune stimulation.
Apart from potential therapeutic and pharmacologic applications of CLA as set forth above, there has been much excitement regarding the use of CLA nutritively as a dietary supplement. CLA has been found to exert a profound generalized effect on body composition, in particular redirecting the partitioning of fat and lean tissue mass. U.S. Pat. No. 5,554,646 (Cook, et al.) discloses a method utilizing CLA as a dietary supplement in which pigs, mice, and humans were fed diets containing 0.5 percent CLA. In each species a significant drop in fat content was observed with a concomitant increase in protein mass. It is interesting that in these animals, increasing the fatty acid content of the diet by addition of CLA resulted in no increase in body weight, but was associated with a redistribution of fat and lean within the body. Another dietary phenomenon of interest is the effect of CLA supplementation on feed conversion. U.S. Pat. No. 5,428,072 (Cook, et al.) provided data showing that incorporation of CLA into animal feed (birds and mammals) increased the efficiency of feed conversion leading to greater weight gain in the CLA supplemented animals. The potential beneficial effects of CLA supplementation for food animal growers is apparent.
In the development of a defined commercial source of CLA for both therapeutic and nutritional applications, a process for generating large amounts of defined material is needed. The problem with most CLA products made by conventional approaches is their heterogeneity, and substantial variation in isoform from batch to batch. Considerable attention has been given to the fact that the ingestion of large amounts of hydrogenated oils and shortenings, instead of animal tallow, has resulted in a diet high in trans-fatty acid content. For example, Holman, et al., PNAS, 88:4830 (1991) showed that rats fed hydrogenated oils gave rise to an accumulation in rat liver of unusual polyunsaturated fatty acid isomers, which appeared to interfere with the normal metabolism of naturally occurring polyunsaturated fatty acids. These concerns were summarized in an early Editorial in Am. J. Public Health, 84: 722 (1974) Therefore, there exists a strong need for a CLA biologically active product of defined composition.
In the typical animal or human diet, most fatty acids are not provided in free fatty acid form, but rather in phosph- or acyl-glyceride form. The general type and distribution of fatty acid containing lipid components in plant tissue is described in detail in Ohlrogge, et al., supra. For many feed and food applications it is desirable to present the fatty acids in their acylglycerol form. The uptake and metabolism pathways and kinetics differ from the acylglyceride and free acid forms. Most importantly, the binding, rheology, and palatability properties of these respective compounds differs. True triacylglycerols are considerably more palatable, with markedly reduced aftertaste.
There are chemical processes which effect the acylation of a glycerol backbone with fatty acids of straight chain structure. Generally, the first and third hydroxyl positions of the glycerol molecule are derivatized first, and finally the second position is acylated. Reaction to completion is difficult, and selection of conditions able to drive the reaction to saturation, result in double bond rearrangements and transacylation events giving rise to a fatty acid moiety content differing from the distribution of the original preparation.
An alternative to chemical methods of forming triacylglycerol is the use of enzymes such as various lipases. It is found that fatty acids or esters derived therefrom, and glycerol are quite efficiently reacted under very mild conditions in the presence of solid phase bound lipases. WO 91/16443 discloses a method utilizing C. antarctica lipase, C. fugosa lipase, and other enzymes to catalyze formation of triacylglycerides from free polyunsaturated fatty acids or their esters and glycerol. Conversion to glycerides is essentially complete at 98 percent when the resulting water or lower polyhydric alcohol byproducts are continuously removed. Maintenance of isomer distribution is also reported in Haraldsson, et al., Tetrahedron 51: 941 (1995). Again, these results are applicable only to the higher polyunsaturated fatty acids and esters. The degree of reaction, and the influence of chain length and double bond portions on enzyme specificity is discussed in detail in Macrae, Biochemical Soc. Trans. 17: 1146 (1989). An overview of the industrial use of lipases is set forth in Vukson, “Industrial Applications of Lipases”, and Kotting, et al., “Lipases and Phospholipases in Organic Synthesis”, in Paul Woolley and Steffen Petersen (eds.), Lipases: Their Structure, Biochemistry & Application (1994). For the use of lipases in transesterifizing fatty acids to alternate glycerol positions, see Haraldsson, et al. JAOCS 74: 1418 (1997).